Assays for measuring enzyme activity are widely employed in the pharmaceutical and environmental sciences. With the advent of combinatorial chemistry and high throughput screening there is a growing need for simple, sensitive and cost-effective assays to screen for potential modulators of enzyme activity.
The enzyme aromatase cytochrome P450 19 A1 (EC 1.14.14.1) is the product of the CYP19 gene, a member of the P450 superfamily of genes. Aromatase catalyses the rate-limiting step in oestrogen biosynthesis, the conversion of C19 androgenic steroids to the corresponding oestrogen (FIG. 1), a reaction termed aromatisation since it converts the Δ4-3-one ring of the androgen to the phenolic A-ring of oestrogen (Ciolino et al., 2000, British Journal of Cancer, 83, 333-337).
Oestrogens are the most important etiological factors in the growth and development of many breast carcinomas in both pre- and post-menopausal women. Breast tumours from post-menopausal women contain high levels of 17β-oestradiol despite the presence of low plasma 17β-oestradiol concentrations. It is now widely accepted that breast tumours can synthesise 17β-oestradiol from adrenal androgen precursors. Synthesis occurs through the aromitisation of androstenedione to oestrone by aromatase, followed by conversion of oestrone to 17β-oestradiol by 17β-hydroxysteroid dehydrogenase type 1 (James et al., 2001, Endocrinology 142, 1497-1505). When measured in-vitro, aromatase activity was found to be higher in breast tumours than in adjacent or healthy fat cells. Furthermore, adipose stromal cells surrounding cancerous cells have been shown to contain higher levels of aromatase mRNA than corresponding cells in non-cancerous areas (Chen et al., 1999, Endocrine-Related Cancer, 6, 149-156). Thus aromatase activity in tumours or surrounding tissue is believed to play a significant role in promoting tumour growth due to local production of oestrogen.
Aromatase offers a key point of intervention in the treatment of breast cancer by reducing the activity and consequently the level of oestrogen synthesised at the site of the tumour. Thus aromatase inhibitors provide significant benefit to many breast cancer patients (James et al., 2001, Endocrinology 142, 1497-1505).
Aromatase is an important enzyme not only from a medical and pharmaceutical viewpoint in the treatment of breast cancer but also from an environmental perspective because inhibitors have been identified as potential environmental toxins, or so called ‘endocrine disrupters’ (Mak et al., 1999, Environmental Health Perspectives, 107, 855-860). The development of a simple, high throughput screening assay to identify modulators and particularly inhibitors of aromatase activity is thus of considerable commercial interest.
Fluorescence Detection Methods
Fluorescence-based assays offer significant advantages over radiochemical, ELISA, antibody and more traditional techniques for measuring enzyme activity in terms of simplicity of handling, sensitivity, cost and ease of automation. Recently there has been considerable interest in the application of fluorescence resonance energy transfer (FRET) assays which involve the use of substrates having donor and quenching acceptors on the same molecule. WO 94/28166, for example, reports the use of such FRET labels attached to a polypeptide substrate which fluoresce more intensely on hydrolysis by a protease.
While FRET techniques offer greater sensitivity and reliability for use in screening assays than simple fluorescent intensity techniques, the substrates are considerably more expensive to prepare and purify due to their complex nature. Thus the preparation of FRET labels is demanding in terms of both analytical and/or purification and material costs. Furthermore the only method for distinguishing conventional fluorescent or FRET labels is by their absorption and emission spectra.
Fluorescence lifetime measurements that may be utilised in the present invention offer significant advantages over conventional fluorescence techniques that are based solely on quantifying fluorescence intensity. Fluorescence lifetime is determined from the same spectrally resolved intensity signal, but is additionally resolved in the temporal domain. Fluorescence lifetime techniques provide greater discrimination because the signal is largely unaffected by ‘background noise’. A further advantage with this technique is that several different events can be measured simultaneously by selecting labels having distinguishable lifetimes, thus enabling multiplexing. In addition, measurements of fluorescence lifetime are unaffected by concentration effects and photobleaching.
Aromatase Assays
Several assay formats have been reported for the measurement of aromatase activity. These can be divided into two categories depending on the use of a ‘natural’ or a surrogate substrate. Detection methodologies have included the use of radioisotopic tracers (e.g. Thompson & Siiteri, 1974, Journal of Biological Chemistry, 249, 5364-5372), fluorescence intensity (Crespi et al., Analytical Biochemistry, 1997, 248, 188-190), enzyme activity (e.g. Chabab et al., 1986, Journal of Steroid Biochemistry, 25, 165-169) and fast liquid chromatography (Fauss & Pyerin, 1993, Analytical Biochemistry, 210, 421-423).
Odum and Ashby (Toxicology Letters (2002), 129, 119-122) describe a radiometric assay for measuring aromatase activity using the ‘tritiated water assay’. The assay quantifies enzyme activity based on the release of 3H as 3H2O from the 1β position of the substrate during aromatisation. A final reaction contained rat ovary microsomes and an NADPH generating system together with the substrate 1β(3H)-androstenedione and potential aromatase inhibitors in dimethyl sulphoxide. Reactions were started by addition of the substrate and were carried out at 37° C. for 30 min. Reactions were stopped by addition of chloroform-methanol and the mixture shaken for 60 s. After removal of the solvent, a suspension of dextran-coated charcoal was added. The mixture was left for 1 h at 4° C., centrifuged and 500 μl of the supernatant added to scintillant and counted in a liquid scintillation counter.
Although this assay has been widely used in the literature (e.g. WO 03/045925) as a means for identifying potential inhibitors it is clearly not amenable to high throughput procedures as it is a labour intensive and time-consuming, requiring radiolabelled substrate.
Crespi et al. (Analytical Biochemistry (1997), 248, 188-190) describe a microtitre plate-based fluorimetric intensity assay that can be used to measure the activity of recombinant human aromatase expressed in insect cells and prepared as microsomes. The assay uses dibenzylfluorescein (DBF) as the substrate and reports a number of IC5-50 values that are in many cases different from reported values. These differences are reportedly due to variation in methodology such as substrate choice and the use of cell based systems. The use of a ‘surrogate’ substrate in this second format may explain why the IC550 differ from the published values.
There is therefore a continued need in the pharmaceutical and environmental sciences for improved fluorescence-based assays for measuring aromatase activity. Such assays may have one or more of the following attributes: homogeneity, high sensitivity, good reliability, robustness, simplicity of use, low cost, ease of automation, label specificity and/or more than one form of detection for distinguishing labelled compounds. Preferably the improved assays display more than one of these features and preferably they display all of these features. The present invention seeks to provide novel reagents and methods for performing such an assay.